Firefly luciferase is an enzyme that emits light by converting ATP, D-luciferin and oxygen to AMP, oxyluciferin and carbon dioxide.
Currently known examples of nucleic acid analysis methods based on detection of emitted light using firefly luciferase include a pyrosequencing method (see, for example, Non-Patent Document 1), a gene polymorphism (SNPs) analysis method using the BAMPER method (see, for example, Non-Patent Document 2), and a gene detection method using a hybridization method (see, for example, Non-Patent Document 3).
These nucleic acid analysis methods are based on the principle of converting pyrophosphoric acid, which is released when a nucleotide is incorporated by complementary chain synthesis, to ATP using an enzyme such as ATP sulfurylase or pyruvate phosphate dikinase (PPDK), and measuring light emitted by the formed ATP by a luciferin-luciferase reaction.
For example, in the case of a pyrosequencing method, a primer is hybridized with single-strand DNA (sample nucleic acid) serving as a template, and four types of deoxynucleotides (dATP, dGTP, dCTP and dTTP) are added one type at a time to the reaction liquid in sequence to synthesize a complementary chain. When the complementary chain has been synthesized, pyrophosphoric acid is formed as the reaction product. This pyrophosphoric acid is then converted to ATP by ATP sulfurylase, and the formed ATP is measured for luminescence by a luciferin-luciferase reaction. Next, an unreactive nucleotide is removed by a liquid phase method in which an unreactive nucleotide is decomposed by apyrase and the like, or by a solid phase method in which a template DNA is bound to a solid phase and then it is washed with the reaction liquid. The sequence of the sample nucleic acid is then determined by repeating this series of steps while changing the nucleotide.
Although the length of a base sequence able to be determined with the pyrosequencing method is only about 100 bases at a time, this method is attracting attention as a method that enables large-scale nucleic acid analyses to be performed at low cost by analyzing in parallel using a picotiter plate having several million wells per plate.
However, in each of the types of nucleic acid analysis methods based on the principle described above, it is frequently necessary to add a reagent in the form of dATP to the reaction liquid to serve as a substrate of the complementary chain synthesis reaction. Moreover, dATP is also frequently contained in the sample nucleic acid. Since dATP has a structure that resembles that of ATP, although it is much weaker than ATP, it acts as a substrate of luciferase resulting in luminescence. Thus, when analyzing nucleic acid in a reaction liquid containing this dATP using a luciferin-luciferase reaction, the presence of the dATP increases the amount of background light emitted, resulting in the problem of a decrease in analysis sensitivity.
A method that uses a derivative of dATP in the form of deoxyadenosine α-thiotriphosphate (dATPαS) as an alternative to ATP has been reported that prevents emission of background light caused by dATP (see, for example, Patent Document 1). However, dATPαS is extremely expensive in comparison with dATP, and has problems such as poor incorporation efficiency into DNA polymerase reactions and preventing the complementary chain synthesis reaction from proceeding properly.
In addition, in a pyrosequencing method, a method for inhibiting emission of background light attributable to dATP by reducing the rate of content of dATP has also been reported (see, for example, Patent Document 2). However, when the rate of content of dATP of dATP is reduced, the complementary chain synthesis is unable to be fully completed due to a shortage of the required amount of dATP in the case where thymine (T) is continued in the sequence of the template DNA. Thus, the amount of emitted signal light does not correspond to the number of thymine, and since unreacted thymine remains, the reaction is unable to proceed to the next sequence resulting in the problem of being unable to accurately determine the base sequence being analyzed.                Patent Document 1: International Publication WO 98/13523        Patent Document 2: Japanese Laid-Open Patent Publication No. 2007-68450        Non-Patent Document 1: Anal. Biochem., 1987, vol. 167, pp. 235-238        Non-Patent Document 2: Nucleic Acids Res., 2001, vol. 29(19), p. 93        Non-Patent Document 3: Anal. Biochem., 2004, vol. 333, pp. 296-302        